RNA interference (RNAi) employing short double-stranded RNA (siRNA) is a powerful tool for silencing gene expression (WO 01/29058, WO 01/68836, WO 01/75164). Large fragments of double-stranded RNA (dsRNA) elicit a non-specific response in mammalian cells through activation of the interferon (IFN) response pathway that leads to suppression of translation and cell death (Yang, et al., Mol. Cell. Biol. 21:7807-7816 (2001) and Wianny, et al., Nat. Cell Biol. 2:70-25, 25-33 (2000)). The standard method for generating siRNA is based on chemical synthesis of a pre-determined short sequence. In addition to the high cost of this method, computer models are generally required to predict the short sequences effective for RNAi experiments.
A mixture of short lengths of dsRNA obtained through partial digestion of large dsRNA with RNase III in the presence of magnesium ion buffer has been shown to “knock-down” the expression of cognate genes in cultured mammalian cell lines via RNAi (Yang, et al., Proc. Nat'l. Acad. Sci. USA 99:9942-9947 (2002)). However, achieving partial digestions yielding the right size range of product is often a difficult and time-consuming process and requires gel separation to obtain fragments of the desired size. Furthermore the inclusion of all possible sequences contained in the starting material is not ensured. US published application US-2004-0038278, herein incorporated by reference, describes how RNase III in the presence of transition metal ions can produce a heterogeneous mixture of fragments of a size suitable for gene silencing. This is a significant improvement on existing methods of making siRNA fragments. However, it would be desirable to increase the flexibility of the methodology for example to permit non-critical incubation times that can vary in a broad range as determined by the convenience of the experimenter and/or to increase the range of buffers that might be used to create a heterogeneous siRNA mixture.